The arcABC operon of Pseudomonas aeruginosu encodes arginine deiminase, catabolic ornithine carbamoyltransferase and carbamate kinase, respectively. We have determined the nucleotide sequences of the arcA and urcC genes. The arcA open reading frame specifies a polypeptide of 46.3 kDa. The same molecular mass was obtained for the subunit of purified arginine deiminase after electrophoresis under denaturing conditions. The N-terminal amino acid sequence of arginine deiminase was in agreement with the corresponding nucleotide sequence. The native arginine deiminase had an estimated molecular mass of 175 -180 kDa, suggesting a tetrametric structure. The enzyme was activated by Mgz+ or Mn2+ and strongly inhibited by Zn". The apparent K,,, for L-arginine was 0.04 mM in the presence of MgZf and 0.47 mM without Mgz+. The arcC open reading frame codes for a 33-kDa protein, confirming the molecular mass previously reported for the subunit of carbamate kinase. The translation-initiation site of arcC was determined by deletion mapping. Two regions of dyad symmetry found between arcA and arcC might stabilize the putative arcABC transcript in the upstream (arcA) region; this might contribute to the high level of arcA expression as compared to the moderate level of arcC expression.Carbamate kinase had 37% sequence similarity (and 13.5% identity) with the C-terminal part of carbamoylphosphate synthetase (large subunit) from Escherichiu coli. Arginine deiminase had no apparent similarity with argininosuccinate lyase. Thus, the arcA and arcC genes do not appear to be closely related to arginine biosynthetic genes, whereas it had previously been shown that the arcB gene has a high degree of identity with the arginine biosynthetic argF genes of P. aeruginosa and E. c o kThe latter part of arginine biosynthesis and the catabolic arginine-deiminase pathway have a number of metabolites in common (Fig. 1). In Pseudomonas ueruginosa the enzymes which catalyze these reactions are all distinct [I, 21. A futile cycle is avoided by several control mechanisms. Carbamoylphosphate synthetase is derepressed by limitation of arginine or pyrimidines, activated by ornithine and inhibited by UMP [3]. The anabolic ornithine carbamoyltransferase is repressed by arginine and forms, with citrulline, a dead-end complex, which prevents the back reaction [4 -61. The three enzymes of the arginine deiminase pathway are coordinately induced by oxygen limitation and by arginine [7]. The catabolic ornithine carbamoyltransferase shows highly cooperative carbamoyl-phosphate binding and hence does not catalyze the anabolic reaction in vivo. AMP, CMP and inorganic phosphate are activators of the enzyme [8, 91 (V.S., unpublished results). Carbamate kinase is inhibited by ATP [lo]. Thus, conditions of energy depletion favour the functioning of the arginine deiminase pathway. Correspondence to D. Haas, Mikrobiologischcs Institut, ETHAhbreviution. X-gal, 5-bromo-4-chloro-3-indolyl-~-~-galactoside. Enzymes. Arginase (EC 3.5.3.1); arginine deiminase (EC 3.5.3.6 The gene...

We have determined the complete nucleotide sequence of the arcB gene from Pseudomonas aeruginosa strain PA0 and we have purified the arcBproduct, the catabolic ornithine carbamoyltransferase (EC 2.1.3.3), to apparent homogeneity from the same strain. The N-terminal amino acid sequence, the total amino acid composition and the subunit size of the purified enzyme were in agreement with nucleotide sequencing results, which predict a polypeptide of 336 amino acids (Mr 38 108). Crosslinking experiments suggest that the native enzyme (apparent M , approx. 420000) basically consists of a trimer aggregating to form nonamers or dodecamers.The arcB gene of P. aeruginosa had strong homology with the argFand arglgenes which code for the anabolic ornithine carbamoyltransferase isoenzymes in Escherichia coli; 63% of the nucleotides and 57% of the amino acids were absolutely conserved in arcB and argF. This indicates a close evolutionary relationship between these genes although their products have different physiological functions in the cell.Under conditions of induction (energy depletion) the catabolic ornithine carbamoyltransferase represented >, 10% of the total cellular protein. Like other highly expressed Pseudomonas genes, the arcB gene was found not to use seven codons which correspond to minor or weakly interacting tRNA species in E. coli.Fluorescent pseudomonads have two ornithine carbamoyltransferases (OTCases) [l, 21. The anabolic enzyme participates in arginine biosynthesis and catalyzes the formation of citrulline and phosphate from ornithine and carbamoyl-phosphate [3]. The catabolic enzyme is involved in arginine degradation via the arginine deiminase pathway and promotes the reverse reaction, i. e. the phosphorolytic cleavage of citrulline, giving ornithine and carbamoylphosphate [l, 41. Both Pseudomonas OTCases are endowed with specific and unique control features that strongly favour the physiological reaction over the back reaction [5, 61. The primary structures of anabolic OTCases from a variety of organisms have been established [7 -91. These enzymes generally are trimers consisting of equal subunits of 35 -40 kDa [lo]. In Escherichia coli the two OTCase isoenzymes, products of the argF and argl genes, are very similar [7]. They also show considerable sequence homology with anabolic OTCases from eukaryotic organisms and with bacterial aspartate carbamoyltransferases [9, 11, 121 that the catabolic OTCase of Pseudomonas putida shares antigenic determinants with the anabolic argF OTCase of E. coli but not with anabolic OTCases from several other bacterial species including P. putida [14]. Thus, in the absence of nucleotide sequence information on catabolic OTCases, it is uncertain whether the catabolic and anabolic OTCases have limited or extended structural similarities.We have recently cloned the arcABC operon specifying the arginine deiminase pathway enzymes from P. aeruginosa [15]. Under conditions of induction, i.e. during limiting aeration and in the presence of arginine, these enzymes are very stro...